CN116769746A - 一种提高毕赤酵母分泌环糊精葡萄糖基转移酶能力的方法 - Google Patents
一种提高毕赤酵母分泌环糊精葡萄糖基转移酶能力的方法 Download PDFInfo
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Abstract
本发明提供了一种提高毕赤酵母分泌环糊精葡萄糖基转移酶能力的方法，属于生物学技术领域，该菌种通过P2A顺反子序列将多个信号肽串联，再连接环糊精葡萄糖基转移酶基因，采用食品安全菌株巴斯德毕赤酵母作为底盘菌进行异源表达，可有效促进了环糊精葡萄糖基转移酶的分泌。本发明显著提高了胞外的蛋白分泌含量，50L发酵罐120h后，发酵液上清蛋白浓度可达10.2g/L，环糊精葡萄糖基转移酶的环化活力可达65.2U/ml，较原始表达系统提高了10倍。
Description
技术领域
本发明涉及分子生物学技术领域,尤其涉及一种提高毕赤酵母分泌环糊精葡萄糖基转移酶能力的方法。
背景技术
巴斯德毕赤酵母(Pichia pastoris)正在被开发为广泛使用的用于重组蛋白生产的宿主生物，优势包括快速生长和高分泌能力。目前已报道的最优信号肽为源于酿酒酵母的α交配因子分泌信号肽，为了更容易的分泌蛋白质以便从细胞外培养基中纯化，提出了通过RA顺反子序列连接α信号肽和DSE4信号肽。通过增强毕赤酵母内外源蛋白途径中的信号识别强度，提高毕赤酵母的蛋白分泌能力。
信号肽可以有效的引导新生肽穿越原核生物的质膜或真核生物的内质网膜。信号肽引导核糖体并定位在内质网的通道上，使得核糖体附着在内质网上，并且不断延长的蛋白质链穿过通道，然后信号肽被剪切，新合成的蛋白质输送至内质网内腔中，蛋白最终通过运输囊泡转运到各种亚细胞器中及细胞外。
已有相关报道，可以通过更换启动子或者信号肽、增加分子伴侣的方法提高毕赤酵母分泌外源蛋白的能力，但是不同的启动子、信号肽或分子伴侣对不同的外源蛋白促进分泌的效果不同，针对环糊精葡萄糖基转移酶，目前未有能够极大促进其分泌的信号肽的相关报道。
本次实验通过验证不同单信号肽，例如α信号肽、DSE4信号肽、DAN4信号肽、GAS1信号肽、MSB2信号肽、FRE2信号肽、EXG1信号肽、SCW11信号肽；和不同双信号肽，例如α+DSE4信号肽、α+DAN4信号肽、α+GAS1信号肽、α+MSB2信号肽、α+FRE2信号肽、α+EXG1信号肽、α+SCW11信号肽等，以期获得一组能够极大促进毕赤酵母分泌糊精葡萄糖基转移酶的信号肽。
发明内容
为了解决毕赤酵母分泌环糊精葡萄糖基转移酶蛋白量低的问题，本发明提供了一种提高毕赤酵母分泌环糊精葡萄糖基转移酶能力的方法，提高环糊精葡萄糖基转移酶在毕赤酵母发酵液上清中的含量。
本发明提供了一种在毕赤酵母中用于分泌环糊精葡萄糖基转移酶的信号肽序列，编码所述信号肽的蛋白序列如SEQ ID NO.1或SEQ ID NO.2所示。
在本发明的一种实施方式中，所述信号肽α信号肽和EXG1信号肽是通过P2A序列串联后形成，新的信号肽如SEQ ID NO.1或SEQ ID NO.2所示。
在本发明的一种实施方式中,新的信号肽与环糊精葡萄糖基转移酶连接，环糊精葡萄糖基转移酶的核苷酸序列如SEQ ID NO.3所示。
本发明还提供了一种携带上述信号肽、环糊精葡萄糖基转移酶的基因的重组载体及含有重组载体的重组毕赤酵母菌株。
本发明还提供了一种重组毕赤酵母菌，所述重组毕赤酵母菌表达了来源于B.circulans strain 251的环糊精葡萄糖基转移酶，编码所述环糊精葡萄糖基转移酶的核苷酸序列如SEQ ID NO.3所示。
在本发明的一种实施方式中，所述重组毕赤酵母菌以Pichia pastoris GS115为表达宿主，以pPIC9K质粒为表达载体。
在本发明的一种实施方式中,利用50L发酵罐发酵所得的发酵液上清中蛋白含量至少为4.2g/L，较对照表达系统提高了16倍；环糊精葡萄糖基转移酶的酶活至少为65.2U/ml，较对照表达系统提高了10倍。
附图说明：
图1:携带含有串联信号肽、环糊葡萄糖基转移酶精基因序列的重组质粒。
具体实施方式
本发明用到了遗传工程和分子生物学领域使用的常规技术和方法﹐以pPIC9K质粒为骨架，将a-factor信号肽、P2A短肽、EXG1信号肽和环糊精葡萄糖基转移酶使用Gibson连接酶50度连接30分钟后，转化大肠杆菌DH5α感受态，获得重组质粒pPIC9K-DS-CGT251。
培养基配方：
大肠杆菌培养基(LB培养基)：0.5％酵母提取物，1％蛋白胨，1％NaCL，pH7.0；
酵母培养基(YPD培养基)：1％酵母提取物、2％蛋白胨、2％葡萄糖；
酵母筛选培养基MD培养基)：2％蛋白胨、2％琼脂糖；
BMGY培养基：2％蛋白胨，1％酵母提取物，100mM磷酸钾缓冲液(pH6.0)，1.34％YNB，4×10-5生物素，1％甘油；
BMMY培养基：2％蛋白胨，1％酵母提取物，100mM磷酸钾缓冲液(pH6.0)，1.34％YNB，4×10-5生物素，0.5％甲醇；
LB-AMP培养基：0.5％酵母提取物，1％蛋白胨，1％NaCL，100ug/mL氨苄青霉素，pH7.0；
LB-AMP平板：0.5％酵母提取物，1％蛋白胀，1％NaCL，1.5％琼脂，100ug/mL氨苄青霉素，pH7.0；
发酵培养基配方：磷酸二氢钾2.2g/L，硫酸镁1.5g/L，磷酸氢二氨4.5g/L，硫酸钙2.3g/L，柠檬酸1.7g/L，补加碳源为甲醇直至发酵结束。
下面结合具体实施方式对本发明进行详细描述。
实施例1α信号肽和EXG1信号肽串联
将来源于Porcine teschovirus的P2A序列和源于Pichia pastoris的DSE4信号肽通过同源重组方式依次连接至pPIC9k载体的α信号肽后面，所述pPIC9k载体自带α信号肽，获得重组载体pPIC9K-DS。使用引物见下表：
按照下述加量和PCR程序，以全合成的P2A序列为模板，使用引物P2A片段-F/R扩增P2A片段；以pPIC9K载体为模板，使用P2A载体-F/R扩增载体序列，PCR程序为98℃2min，98℃30s，60℃30s，30个循环，72℃10min：
表1PCR反应体系加量
按照下述方法进行同源重组：
PCR程序为50℃30min：
| 成分 | 用量 |
| 同源重组酶 | 5μL |
| P2A片段 | 1μL |
| pPIC9k载体片段 | 2μL |
| ddH2O | 2μL |
| Total | 10μL |
表2同源重组反应体系加量
转化体系全部加入DH5α感受态细胞，冰浴30min，42℃90s，加入5ml LB液体培养基，37℃150rpm/min 90min，完全铺于含有100mg/ml的遗传霉素的LB固体平板上，16h后挑取单克隆菌落，扩培后获得大量重组质粒。
表3引物序列表
实施例2环糊精葡萄糖基转移酶基因的克隆及载体构建
将来源于B.circulans strain 251的环糊精葡萄糖基转移酶基因命名为CGT251,其编码的氨基酸序列为SEQ ID NO:3。使用同源重组方式连接至pPIC9K-DS载体上，获得重组载体pPIC9K-DS-CGT251。引物序列如下：
方法与实施例1相同
实施例3对照载体的构建
将来源于B.circulans strain 251的环糊精葡萄糖基转移酶基因命名为CGT251,其编码的氨基酸序列为SEQ ID NO:3。使用同源重组方式连接至pPIC9K载体上，获得重组载体pPIC9K-CGT251。引物序列如下:
方法与实施例1相同
环糊精葡萄糖基转移酶的重组表达
重组酵母表达质粒pPIC9K-CGT251/pPIC9K-DS-CGT251用下列引物进行线性化，表达质粒线性化片段通过电穿孔法转化毕赤酵母GS115，在MD平板上筛选得到毕赤酵母重组菌株GS115/pPIC9K-CGT，筛选单拷贝的转化子。
分别挑取单个转化子扩培出1LYPD摇瓶种子液，转接于50L发酵罐中，27℃、300rpm发酵培养120h后,获得发酵上清液，离心去除菌体,得发酵上清液。采用下述方法分别测定各转化子发酵上清液中蛋白质含量和环糊精葡萄糖基转移酶的活力。结果显示,所述对照组转化子中发酵蛋白浓度最高达到1.01g/L，酶活最高的达到6.4U/ml；实验组转化子中发酵蛋白浓度最高达到10.2g/L，酶活最高的达到65.2U/ml，为出发菌株的10倍，申请人将实验组转化子命名为毕赤酵母CGT-DS1(Pichia pastoris CGT-DS1)。
(1)蛋白浓度的测定原理
考马斯亮蓝(Coomassie Brilliant Blue)法测定蛋白质浓度，是利用蛋白质-染料结合的原理，定量的测定微量蛋白浓度的快速、灵敏的方法。这种蛋白质测定法具有超过其他几种方法的突出优点，因而正在得到广泛的应用。这一方法是目前灵敏度最高的蛋白质测定法；考马斯亮蓝G-250染料，在酸性溶液中与蛋白质结合，使染料的最大吸收峰(max)的位置，由465nm变为595nm，溶液的颜色也由棕黑色变为蓝色。通过测定595nm处光吸收的增加量可知与其结合蛋白质的量。研究发现，染料主要是与蛋白质中的碱性氨基酸(特别是精氨酸)和芳香族氨基酸残基相结合。
(2)蛋白浓度的测定方法
考马斯亮蓝G-250100mg溶于50mL 95％乙醇中，加入100mL85％磷酸,用蒸馏水稀释至1000mL；结晶牛血清蛋白，预先经微量凯氏定氮法测定蛋白氮含量，根据其纯度用纯水配制成Img/mL蛋白溶液。
蛋白浓度标准曲线
取7支试管，按下表平行操作：
摇匀，1h内以0号管为空白对照，在595nm处比色，以A595nm为纵坐标，标准蛋白含量为横坐标，在坐标纸上绘制标准曲线。
发酵液样品蛋白质浓度测定
测定方法同上，取合适的未知样品体积，使其测定值在标准曲线的直线范围内。根据所测定的A595nm值，在标准曲线上查出其相当于标准蛋白的量，从而计算出未知样品的蛋白质浓度(mg/mL)。
(3)环糊精葡萄糖基转移酶酶活单位的定义
环糊精葡萄糖基转移酶结合酚酞生成络合物，能够在碱性溶液中包合酚酞使得原本的紫红色溶液吸光度降低，通过分光光度计在550nm处测定吸光值，可以通过计算吸光度较空白的降低值来计算环糊精葡萄糖基转移酶的含量，吸光度降低程度与β-CD的浓度在一定范围内呈线性关系。一个酶活单位定义为标准条件下每分钟将淀粉转化为1μmolβ-CD所需要酶的量。
(4)环糊精葡萄糖基转移酶酶活测定
取0.9mL的4％可溶性淀粉溶液(溶于65mmol/L，pH 6.0PBS缓冲液)，加入0.1mL适当稀释的酶液，充分混合后于50℃反应20min后，加入3.5mL 30mmol/L的NaOH溶液终止反应，再加入0.5mL 0.02％的酚酞显色液(溶于5mmol/L的Na2CO3溶液)显色15min，于550nm处测定其吸光度。
酶活力(U/mL)＝(a-b)/A x 1000x稀释倍数
注:式中a为对照组OD值,b为样品OD值
序列表
>SEQ ID NO.1
MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKREAEAATNFSLLKQAGDVEENPGPMSFSSNVPQLFLLLVLLTNIVSG
>SEQ ID NO.2
MSFSSNVPQLFLLLVLLTNIVSGATNFSLLKQAGDVEENPGPMRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKREAEA
>SEQ ID NO.3
APDTSVSNKQNFSTDVIYQIFTDRFSDGNPANNPTGAAFDGTCTNLRLYCGGDWQGIINKINDGYLTGMGVTAIWISQPVENIYSIINYSGVNNTAYHGYWARDFKKTNPAYGTIADFQNLIAAAHAKNIKVIIDFAPNHTSPASSDQPSFAENGRLYDNGTLLGGYTNDTQNLFHHNGGTDFSTTENGIYKNLYDLADLNHNNSTVDVYLKDAIKMWLDLGIDGIRMDAVKHMPFGWQKSFMAAVNNYKPVFTFGEWFLGVNEVSPENHKFANESGMSLLDFRFAQKVRQVFRDNTDNMYGLKAMLEGSAADYAQVDDQVTFIDNHDMERFHASNANRRKLEQALAFTLTSRGVPAIYYGTEQYMSGGTDPDNRARIPSFSTSTTAYQVIQKLAPLRKCNPAIAYGSTQERWINNDVLIYERKFGSNVAVVAVNRNLNAPASISGLVTSLPQGSYNDVLGGLLNGNTLSVGSGGAASNFTLAAGGTAVWQYTAATATPTIGHVGPMMAKPGVTITIDGRGFGSSKGTVYFGTTAVSGADITSWEDTQIKVKIPAVAGGNYNIKVANAAGTASNVYDNFEVLSGDQVSVRFVVNNATTALGQNVYLTGSVSELGNWDPAKAIGPMYNQVVYQYPNWYYDVSVPAGKTIEFKFLKKQGSTVTWEGGSNHTFTAPSSGTATINVNWQP 。
Claims (3)
1.一种提高毕赤酵母分泌环糊精葡萄糖基转移酶能力的方法，其特征在于：通过P2A序列串联后形成信号肽α信号肽和EXG1信号肽，所述信号肽的核苷酸序列如SEQ ID NO.1或SEQ ID NO.2所示。
2.如权利要求1所述的提高毕赤酵母分泌环糊精葡萄糖基转移酶能力的方法，其特征在于，所述信号肽与环糊精葡萄糖基转移酶连接，环糊精葡萄糖基转移酶的核苷酸序列如SEQ ID NO.3所示。
3.如权利要求1所述的提高毕赤酵母分泌环糊精葡萄糖基转移酶能力的方法，所述毕赤酵母菌以PichiapastorisGS115为表达宿主，以pPIC9K质粒为表达载体。
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